Modern neuroscience seeks to identify, and manipulate with precision, specific cell types in the brain to identify mechanisms of circuit assembly, function, and neural disease. These technologies are a prerequisite to a large number of fields of study, from systems neuroscience to molecular developmental analysis, and are key to providing insights not only into neural function, but also disease processes, including aging, neurodegeneration, and neural repair developing tools to label and manipulate specific neuronal types is generally costly and time-consuming. The objective of this application is to develop and release to the scientific community an extensive and versatile toolkit for manipulation of the nervous system of Drosophila, a powerful genetic model that has generated many fundamental insights into nervous system function and development. This application has three central aims. First, we propose to generate 4,000 new transgenic lines based on the recently developed InSITE system. These strains will allow cell-specific manipulations of gene expression in the vast majority of neurons in the fly. Each of these insertion sites will be molecularly mapped, and the expression pattern captured by each line will be described in both larval and adult fly brains. Second, using this collection, we will derive cell-type specific intersectional tools, as well as independent transcriptional regulatory systems, allowing gene expression to be manipulated in multiple individual cell types, independently. Third, we will create a database describing these data that will enable the community to make full use of this resource to address a wide range of scientific questions. This collection will provide a platform for introducing a versatile collection of markers and drivers of gene expression into a large number of the neuronal types present in the larval and adult nervous systems for release to the Drosophila community. Once this resource is established, the methods for using and extending the collection involve only basic genetic crosses that can be adapted in any lab. We expect that this collection will seed an expanding, highly versatile, and cost-effective collection of tools fo manipulation of the nervous system and gene discovery.